Validation of candidate bovine reference genes for use with real-time PCR

Accurate quantification with real-time PCR requires the use of stable endogenous controls. Recently, there has been much debate concerning the stability of commonly used reference or housekeeping genes. To address this concern, a number of statistical approaches have been designed to analyse data and assist in determining the most appropriate reference genes for experimental comparisons. In this study, three programs, BestKeeper, Norm Finder, and geNorm were used to assess four candidate reference genes: 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), acidic ribosomal protein large (RPLP0) and β-actin, for use in expression profiling of individuals from divergent cattle genotypes subject to parasitic challenge with the cattle tick Boophilus microplus. Results demonstrated β-actin and GAPDH were the most suitable reference genes in blood and could be used either individually or combined as an index to normalise data. RPLP0 was identified as the least stable gene, while 18S rRNA was omitted as being too highly expressed. As the recommendations on the most suitable reference genes varied between the programs, it is recommended that more than one should be utilised, to ensure the most robust experimental tools are selected.